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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24. April 2002
Deviations:
yes
Remarks:
relative humidity in animal roon was 26-65% for a maximum of 5 hours
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): C-SAT 090069
- Analytical purity: 89-91% active substance
- Physical state: amber coloured viscous liquid
- Lot/batch No.: C-081
- Mol. weight: 263 g/mol
- Expiration date of the lot/batch: Feb 26, 2013
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: harlan Laboratories B.V, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.4 - 22.5g
- Housing: single caging
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 25-65%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25, 50%
No. of animals per dose:
4
Details on study design:
In order to study a possible allergenic potential of the test substance, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled
thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight.
The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a -scintillation counter.

Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 50 % solution in acetone:olive oil (4+1) after long vortexing.
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 25 and 50% each on three consecutive days.
In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 10, 25, and 50%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

Test Item Preparation
The test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1) was quantitatively added. The different test item concentrations were prepared serially.
The preparations were made freshly before each dosing occasion.
The dose calculation was adjusted to purity (a correction factor of 1.1 was used).

Experimental Design and Procedures
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 10, 25, and 50% (w/w) in acetone:olive oil (4+1). The application volume, 25 Ul, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone
(control animals).

Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 Ul of 79.7 UCi/ml 3HTdR (corresponds to 19.9 UCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Ear Thickness
Prior to the first application of the test item and prior to treatment with 3HTdR the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, 30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 Um mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
The ANOVA (Dunnett-Test) was conducted on the values determined for the ear thickness to assess wether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were conducted together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
12.8
Test group / Remarks:
test group 2, 10% test item concentration
Parameter:
SI
Value:
16.1
Test group / Remarks:
test group 3, 25% test item concentration
Parameter:
SI
Value:
15.74
Test group / Remarks:
test group 4, 50% test item concentration
Parameter:
other: disintegrations per minute (DPM)
Value:
16
Test group / Remarks:
background 1, 1 ml 5% trichloroacetic acid
Parameter:
other: disintegrations per minute (DPM)
Value:
15
Test group / Remarks:
background 2, 1 ml 5% trichloroacetic acid
Parameter:
other: disintegrations per minute (DPM)
Value:
3 821
Test group / Remarks:
control group
Parameter:
other: disintegrations per minute (DPM)
Value:
48 735
Test group / Remarks:
test group 1, 10% test item concentration
Parameter:
other: disintegrations per minute (DPM)
Value:
61 276
Test group / Remarks:
test group 2, 25% test item concentration
Parameter:
other: disintegrations per minute (DPM)
Value:
59 904
Test group / Remarks:
test group 3, 50% test item concentration
Parameter:
EC3
Test group / Remarks:
not calculated, SI above 3
Remarks on result:
not determinable

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were

observed during the study period.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to

treatment with 3HTdR, was within the range commonly recorded for animals of this strain

and age.

Ear Thickness

The measured ear thickness of all animals treated was recorded prior to the 1st application

and prior to necropsy. A statistically significant increase in ear thickness was observed for

the animals treated with 10 and 25% test item concentration (p<0.001).

Discussion

In order to study a possible contact allergenic potential of the test substance, three groups

each of four female mice were treated daily with the test item at concentrations of 10, 25,

and 50% (w/w) in acetone:olive oil (4+1) by topical application to the dorsum of each ear

(left and right) for three consecutive days. A control group of four mice was treated with

the vehicle (acetone:olive oil (4+1)) only. Five days after the first topical application the

mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl

thymidine). Approximately five hours after intravenous injection, the mice were sacrificed,

the draining auricular lymph nodes excised and pooled per group. Single cell suspensions

of lymph node cells were prepared from pooled lymph nodes, which were subsequently

washed and incubated with trichloroacetic acid overnight. The proliferative capacity of

pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine

measured in a scintillation counter.

All treated animals survived the scheduled study period and no signs of local irritation or

systemic toxicity were observed. A statistically significant increase in ear thickness was

observed in the animals treated with 10 and 25% test item concentration (p<0.001).

However, this was not seen in the animals treated with 50% test item concentration.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test

concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared

with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated

concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 12.80, 16.10, and 15.74 were determined with the test

item at concentrations of 10, 25, and 50% in acetone:olive oil (4+1). The EC3 value could

not be calculated, since all obtained SI´s were above 3.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In a Local Lymph Node Assay according to OECD Test Guideline 429 under GLP, the substance was determined to be skin sensitising.
Executive summary:

The skin sensitisation potential of the test substance was inverstigated in a Local Lymph Node Assay. Three groups of four female mice were treated by topical application to the dorsum of each ear daily for three days with the concentrations 10, 25 and 50%. Acetone:olive oil (4 +1) was used as vehicle. Five days after study initiation, the mice were injected intravenously into a tail vein with radio-labbeld thymidine and sacrificied five hours later.

No mortality occured and no signs of local irritation or systemic toxicity were present. Stimulation indexes were 12.80, 16.10 and 15.74, all being larger than the cut-off of 3. Consequently, the test substance is considered to be a skin sensitiser.